An oligonucleotide synthesis evaluation was begun by the Nucleic Acids Committee last spring to answer two questions: (1) how well are facilities preparing short and intermediate-length oligonucleotides; and (2) how well suited are the products for use as DNA sequencing primers? In addition, a few basic questions concerning instrumen-tation and charges were also included. Laboratories were asked to submit specific 25- and 50-base oligonucleotides, made according to their method of choice.
A total of 72 different laboratories contributed samples to this study. We received 105 25-mers and 103 50-mers for analysis. Samples were synthesized using either Applied Biosystems (80%), Perceptive Biosystems (15%), Beckman (2%), Eppendorf (1%), or Pharmacia (1%) instrumentation. As of May, 1995 the average charge for synthesizing a 25-base long oligonucleotide on a 40-50 nmol scale was $42.00 ($1.68/base). The average charge for a 200 nmol scale synthesis was $69.67 ($2.79/base).
The committee asked study participants to submit unpurified oligonucleotides, but participants were also asked the type of purification they would recommend for the 25-mer if it were to be used as a DNA sequencing primer. Interestingly, the opinions of our membership were quite divided. A majority believed that no purification at all would suffice (41%), while a slightly smaller group (36%) felt that purification by OPC or PAGE was required. Most of the remainder felt that desalting would be adequate (22%). Hopefully, our evaluation of the 105 unpurified samples as sequencing primers under identical conditions will provide useful information on this issue.
To date, each sample has been quantified by UV absorbance, checked by polyacrylamide gel electrophoresis, analyzed by capillary electrophoresis, and tested for performance as a DNA sequencing primer (25-mers only). In general, the capillary electrophoresis analyses showed that the overall quality of oligonucleotides prepared by our members was quite good. However, the performance of the unpurified sequencing primers cannot be assessed until further computerized data analysis. We hope to be able to return individual capillary electrophoresis and automated sequencing chromatograms to study participants and provide more information later this year.
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